Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products

Abstract

In Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non-PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS(-) strain. The W3110 PTS(-), designated VH32, showed limited growth on glucose with a specific growth rate (mu) of 0.03 h(-1). A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc-derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild-type trc promoter resulted in a mu of 0.55 h(-1), corresponding to 89% of the mu measured for W3110 (0.62 h(-1)). In contrast, no increase in specific growth rate (mu) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a mu value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594.

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

“…As glucose uptake via the PTS is known to consume half of PEP produced from glucose and produces PYR (Hernandez-Montalvo et al 2003), an attempt was made to use an alternative pathway for the cells to import glucose. Unlike the PTS which uses PEP as phosphate donor, the GGS transports and phosphorylates glucose using ATP as phosphate donor, thus increasing intracellular PEP and increasing the availability of intracellular GAP.…”

“…Thus, it was hypothesized that the deletion of the PTS could potentially redirect more carbon flux from the central pathway to the synthesis of isoprenoids through the DXP pathway. In E. coli, the PTS is the main glucose transport system and is also responsible for carbon catabolite repression (CCR) and chemotaxis (Hernandez-Montalvo et al 2003). Thus, deletion of the PTS is also pleiotropic and its mutant has several distinct characteristics over the wild-type strain, such as enhancement of intracellular PEP concentration, coexistence of glycolytic and gluconeogenic pathways, and the simultaneous utilization of different carbon sources (Flores et al 2002).…”

“…Transcriptome analysis in these fast-growing strains indicated that the expressions of gene galP (coding for galactose permease), glk (coding for glucose kinase), and pgi (coding for phosphoglucose isomerase) were increased as compared to the wild-type strain, suggesting that these genes were important in enhancing glucose uptake Flores et al 2002). Hence, it was reported that the overexpression of galP and glk enhanced the growth rate of microbes in glucose medium Lu et al 2012) and improved the yields of valuable products such as aromatics (Yi et al 2003), recombinant protein (De Anda et al 2006), and ethanol (Hernandez-Montalvo et al 2003). Combinatorial modulation of galP and glk gene expressions has been used to improve cell growth rate (Lu et al 2012), but it is unclear if such an approach will increase the production of isoprenoids.…”

Biological research survey for the efficient conversion of biomass to biofuels.

“…This leads to sequential sugar utilization, which is disadvantageous for industrial processes to produce ethanol from lignocellulosic materials, as hydrolysis of hemicellulose and cellulose produces C5 sugars such as xylose and arabinose along with glucose. In particular, glucose exerts a repressive effect on the consumption of other sugars, both PTS sugars and non-PTS sugars (24). However, the phenomenon is not restricted to glucose, as many PTS carbohydrates are used in preference over other carbon sources.…”

“…The PTS is a complex system deeply integrated into the cell's physiology and much work is needed to fully understand the results of these modifications. Nevertheless, significant improvements in mixed sugar utilization have already been demonstrated (24)(25)(26)(27).…”